Serum adiponectin quantitative detection reagent, preparation thereof and application of serum adiponectin quantitative detection reagent in ADPN detection

Abstract

The invention provides a serum adiponectin quantitative detection reagent, preparation thereof and application of the serum adiponectin quantitative detection reagent in ADPN detection, and belongs to the technical field of in-vitro detection. The invention provides a serum adiponectin latex immunoturbidimetric reagent which comprises a solution containing ADPN antibody protein functionalized latex microspheres, and the solution containing the ADPN antibody protein functionalized latex microspheres comprises the ADPN antibody protein functionalized latex microspheres, a stabilizer, a protective agent, inorganic salt and a preservative. The preparation process of the reagent adopts a homogeneous phase process, the steps of high-speed refrigerated centrifugation, washing and the like are omitted, the preparation process is simple, and the obtained kit is low in detection lower limit, high in accuracy and high in stability.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostic technology, and relates to serum adiponectin, specifically to a quantitative detection reagent for serum adiponectin, its preparation, and its application in ADPN detection. Background Technology

[0002] Adiponectin (ADPN) is an endogenous bioactive polypeptide and protein secreted by adipocytes. It exists stably in plasma, accounting for approximately 0.01% of plasma proteins. Adiponectin was first discovered in human subcutaneous adipose tissue, plasma, and adipocytes of rodents. Further research has revealed that adiponectin is an insulin-sensitizing hormone that can improve insulin resistance and atherosclerosis. Adiponectin levels can predict the development of type II diabetes and coronary heart disease, and clinical trials have shown a correlation with obesity, diabetes, insulin resistance, atherosclerosis, and inflammation. Therefore, detecting serum adiponectin levels is of great significance for the diagnosis and prognosis of these diseases. Currently, the main methods for determining adiponectin (ADPN) include chemiluminescent immunoassay (CLIA), enzyme-linked immunosorbent assay (ELISA), and latex-enhanced immunoturbidimetry. Chemiluminescence immunoassay offers high sensitivity but requires specialized and expensive equipment. ELISA is extremely cumbersome and complex, with long detection times and poor repeatability. Latex-enhanced immunoturbidimetry utilizes the binding of surface-crosslinked monoclonal or polyclonal antibodies from latex particles of a specific particle size to the antigen to detect its concentration. Existing latex-enhanced immunoturbidimetry methods offer high sensitivity but suffer from poor interference resistance, poor repeatability in the low-value region, and complex preparation processes that are resource-intensive. Currently, commercially available kits for detecting ADPN in humans primarily employ latex-enhanced immunoturbidimetry. The plasma concentration of adiponectin dictates the use of small-diameter microspheres (100-150 nm), but microspheres within this size range cannot fully meet the required low-value sensitivity. Conversely, while using large-diameter microspheres can significantly improve reagent sensitivity, it cannot achieve a superior detection linear range, ultimately resulting in existing reagent kits struggling to balance low-value precision and linear range. Furthermore, due to the easy degradation and poor stability of conventional recombinant adiponectin polymers, most manufacturers on the market use lyophilized powder for calibrator preservation, further increasing reagent preparation costs and process complexity.

[0003] Conventional latex-enhanced immunoturbidimetric assay kits require high-speed centrifugation (18000 r / min) and precipitation, which are complex, costly, and yield-low, constituting a heterogeneous process. In contrast, homogeneous methods omit high-speed refrigerated centrifugation and washing, offering advantages such as simpler operation, lower cost, and higher yield. This implies that EDC, Sulfo-NHS, and uncrosslinked antibodies used in the reaction will coexist in the functional latex microsphere solution. EDC and Sulfo-NHS are chemically unstable and can rapidly degrade in solution. [1] However, when uncrosslinked antibodies are used in assays to detect the target analyte, they compete with the crosslinked antibodies on the functional microspheres for the analyte, leading to a decrease in the detected absorbance value and causing deviation in the results. [2] Meanwhile, the absorbance values ​​of each calibration reaction in the homogeneous method are all lower than those in the heterogeneous method to varying degrees. How to use homogeneous technology to remove uncrosslinked antibodies in the solution or reduce the reactivity of uncrosslinked antibodies remains an urgent problem to be solved.

[0004] Chinese patent CN114295840B discloses a highly sensitive kit for the quantitative determination of adiponectin, comprising reagent R1 and reagent R2. Reagent R2 includes latex microspheres coated with adiponectin monoclonal antibody. The preparation process of the latex microspheres coated with adiponectin monoclonal antibody includes the simultaneous addition of BSA during the labeling process of the latex microspheres and the adiponectin monoclonal antibody. By using large-particle-size latex microspheres, the technical problem of low precision at low values ​​in adiponectin detection kits is improved. However, the preparation process of this kit requires centrifugation and washing, making the preparation process complex.

[0005] Chinese patent CN114295842B discloses an adiponectin detection kit and its preparation method. The kit includes reagent R1 and reagent R2. Reagent R1 comprises buffer, electrolyte, fusion accelerator, surfactant, and preservative. Reagent R2 comprises anti-adiponectin monoclonal antibody-coated latex particles, buffer, protectant, stabilizer, preservative, and surfactant. The surfactant is selected from one or two of Tetronic 1307 and castor oil polyoxyethylene ether. This kit enhances the resistance to interference from specific samples such as chylous and lipophilic samples by using a novel surfactant combination in conjunction with other components, reducing interference from non-specific adsorption. However, the detection limit and accuracy of this kit are relatively poor.

[0006] Chinese patent CN119104740A discloses an improved latex-enhanced immunoturbidimetric assay kit for detecting adiponectin. The kit employs a combination of physical adsorption and chemical coupling during antibody-microsphere conjugation, with physical adsorption being the primary method. However, this kit exhibits poor detection limits and accuracy.

[0007] References: [1] Helen Stowe,1 David Lawrence,1 David J. Newman, et al. AnalyticalPerformance of a Particle-enhanced Nephelometric Immunoassay for SerumCystatin C Using Rate Analysis [J]. Clinical Chemistry, 2001,47(8): 1482-1485. [2] Wang Yishan, Li Daqian, Cui Weiwei, et al. Performance evaluation of immunoturbidimetric assay for adiponectin detection [J]. Labeled Immunoassay and Clinical, 2022, 29(4): 705-710. Summary of the Invention This invention addresses the problem of existing adiponectin detection kits lacking simple preparation processes, low detection limits, and high accuracy. It provides a serum adiponectin latex immunoturbidimetric reagent comprising a solution of ADPN antibody-functionalized latex microspheres. The solution includes ADPN antibody-functionalized latex microspheres, stabilizers, protectants, inorganic salts, and preservatives. This reagent is prepared using a homogeneous process, eliminating steps such as high-speed refrigerated centrifugation and washing, resulting in a simple preparation process and a kit with a low detection limit, high accuracy, and high stability.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: On one hand, the present invention provides a serum adiponectin latex immunoturbidimetric reagent, comprising a solution containing ADPN antibody protein functionalized latex microspheres, wherein the solution containing ADPN antibody protein functionalized latex microspheres comprises ADPN antibody protein functionalized latex microspheres, stabilizers, protectants, inorganic salts and preservatives; The method for preparing the solution containing ADPN antibody protein-functionalized latex microspheres includes the following steps: S1. Take 8%-12% latex microsphere suspension and add 0.20-0.50 mL of buffer A to obtain solution 1; S2. Add 0.02-0.10 mL of buffer A containing 12-21 mg / mL Sulfo-NHS and 0.02-0.10 mL of buffer A containing 3-6 mg / mL EDC to solution 1 to carry out the activation reaction and obtain solution 2. S3. Add 4.0-10.0 mL of buffer B to solution 2, add ADPN antibody, and carry out the antibody-microsphere coupling reaction to obtain solution 3; S4. Add 0-3 mL of buffer B, 0.05-0.30 mL of buffer B containing 10.0-17.5 g / L glycine, and 0.05-0.30 mL of buffer B containing 150-350 g / L BSA to solution 3 to carry out the blocking reaction and obtain solution 4. S5. Add inorganic salts, protective agents, stabilizers, and preservatives to solution 4 and carry out an aging reaction to obtain a solution containing ADPN antibody protein functionalized latex microspheres. The buffer A is selected from any one or more of phosphate buffer, MES buffer, MOPS buffer, glycine buffer, Tris buffer or HEPES buffer; The buffer B is selected from any one or more of phosphate buffer, MES buffer, MOPS buffer, glycine buffer, Tris buffer, or HEPES buffer.

[0009] Preferably, the preparation method includes the following steps: S1. Take 8%-12% of 90-110nm latex microsphere suspension, add 0.20-0.50 mL of buffer A, and shake well at 35-40℃ to obtain solution 1; S2. Under stirring, add 0.02-0.10 mL of buffer A containing 12-21 mg / mL Sulfo-NHS and 0.02-0.10 mL of buffer A containing 3-6 mg / mL EDC to solution 1, and carry out the activation reaction for 10-30 min to obtain solution 2; S3. Under stirring, add 4.0-10.0 mL of buffer B and 0.3-0.6 mg of ADPN antibody to solution 2, and carry out the antibody-microsphere coupling reaction for 2-5 hours to obtain solution 3. S4. Under stirring, add 1-3 mL of buffer B, 0.05-0.30 mL of buffer B containing 10.0-17.5 g / L glycine, and 0.05-0.30 mL of buffer B containing 150-350 g / L BSA to solution 3, and carry out the blocking reaction for 1-3 hours to obtain solution 4. S5. Add inorganic salts, protective agents, stabilizers, and preservatives to solution 4, adjust the pH to 7.0-9.0, and carry out an aging reaction for 24-72 hours to obtain a solution containing ADPN antibody protein functionalized latex microspheres.

[0010] Preferably, the preparation method includes the following steps: S1. Take 10% of 100nm latex microsphere suspension, add 0.25 mL of buffer A, and shake well at 37℃ to obtain solution 1; S2. Under stirring, add 0.04 mL of buffer A containing 12-21 mg / mL Sulfo-NHS and 0.04 mL of buffer A containing 3-6 mg / mL EDC to solution 1, and carry out the activation reaction for 15 min to obtain solution 2; S3. Under stirring, add 6.0 mL of buffer B to solution 2, add 0.3-0.6 mg of ADPN antibody, and carry out the antibody-microsphere coupling reaction for 2-5 hours to obtain solution 3; S4. Under stirring, add 2 mL of buffer B, 0.10 mL of buffer B containing 10.0-17.5 g / L glycine and 0.10 mL of buffer B containing 150-350 g / L BSA to solution 3, and carry out the blocking reaction for 1 hour to obtain solution 4. S5. Add inorganic salts, protective agents, stabilizers, and preservatives to solution 4, adjust the pH to 8.0, and carry out an aging reaction for 24 hours to obtain a solution containing 1.5-3.0 g / mL ADPN antibody protein functionalized latex microspheres.

[0011] Preferably, The buffer A mentioned in step S1 is a 0.1-0.3 mol / L MES buffer, preferably a 0.2 mol / L MES buffer; In step S2, the concentration of Sulfo-NHS in buffer A is 15 mg / mL, and the concentration of EDC in buffer A is 4.5 mg / mL. In step S3, the buffer solution B is a 0.05-0.20 mol / L phosphate buffer, preferably a 0.10 mol / L phosphate buffer; In step S3, 0.4 mg of ADPN antibody was added, and the coupling reaction was carried out on a shaker for 3 hours. In step S4, the concentration of glycine in buffer B is 12.5 g / L, and the concentration of BSA in buffer B is 200 g / L. The final concentration of the ADPN antibody protein-functionalized latex microspheres in solution in step S5 is 2.5 g / mL.

[0012] Preferably, The protective agent is selected from any one or more of BSA, mannitol, chitosan or casein, preferably BSA; The stabilizer is selected from any one or more of sucrose or trehalose; The inorganic salt is selected from any one or more of magnesium chloride, sodium chloride, potassium chloride, calcium chloride, and aluminum chloride, with sodium chloride being preferred; The preservative is selected from any one or more of Proclin 300, sodium azide, phenol, gentamicin, or thimerosal, preferably Proclin 300.

[0013] Preferably, The concentration of the protective agent is 5-100 g / L, the concentration of the stabilizer is 1.0-20.0 g / L, the concentration of the inorganic salt is 1.0-20.0 g / L, and the concentration of the preservative is 0.01-3.0 g / L.

[0014] Preferably, the serum adiponectin latex immunoturbidimetric reagent further includes reagent 1, which includes buffer, surfactant, coagulant, inorganic salt and preservative; The buffer solution is selected from any one or more of citrate buffer, phosphate buffer, carbonate buffer, glycine buffer, Tris buffer or MES buffer, preferably phosphate buffer; The surfactant is selected from any one or more of Tween-20, Tween-80 or Triton X-100, preferably Tween-20; The coagulant is selected from any one or more of polyethylene glycol 8000, polyethylene glycol 6000, and polyethylene glycol 2000, preferably PEG6000; The inorganic salt is selected from any one or more of magnesium chloride, sodium chloride, potassium chloride, calcium chloride, and aluminum chloride, with sodium chloride being preferred; The preservative is selected from any one or more of Proclin 300, sodium azide, phenol, gentamicin, and thimerosal, preferably Proclin 300 (abbreviated as PC-300).

[0015] Preferably, The concentration of the buffer solution is 10-300 mM, the concentration of the surfactant is 0.2-20.0 g / L, the concentration of the coagulant is 0.5-5.0 g / L, the concentration of the inorganic salt is 10-50 g / L, and the concentration of the preservative is 0.01-3.00 g / L.

[0016] On the other hand, the present invention provides the application of the above-mentioned serum adiponectin latex immunoturbidimetric reagent in ADPN detection.

[0017] On the other hand, the present invention provides a method for detecting ADPN, wherein the method is to use the above-mentioned serum adiponectin latex immunoturbidimetric reagent for detection.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The reagent preparation process provided by this invention adopts a homogeneous process, which omits steps such as high-speed refrigerated centrifugation and washing. The preparation process is simple, and the resulting reagent kit has a low detection limit, high accuracy, and high stability. Attached Figure Description

[0019] Figure 1 This is a standard curve plot for the linear range test in the verification example. Detailed Implementation

[0020] Unless otherwise specified, all raw materials and reagents used in this invention were purchased from commercial suppliers, and experiments were conducted in accordance with the operating instructions. Unless otherwise specified, all instruments, equipment, and apparatus used in this invention are conventional instruments, equipment, and apparatus, and experiments were conducted in accordance with the operating instructions and the accompanying reagents.

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention.

[0022] Data analysis and statistical analysis were performed using professional data processing software. One-way ANOVA was used for significance analysis, and P<0.05 was considered to indicate a significant difference.

[0023] The main materials and instruments involved in this invention are described in Table 1.

[0024] Table 1. Material and Instrument Specifications

[0025] Example 1: Preparation and assay method of the reagent kit 1. Preparation of reagent 1 (R1): Place Na₂HPO₄ and KH₂PO₄ in a beaker, add 95% purified water (the total volume of reagent 1), and stir at 120 rpm for 20 min until fully dissolved. Adjust the pH to 7.50 with 3 mol / L potassium hydroxide solution. Add Tween-80, polyethylene glycol 6000, sodium chloride, and preservative PC-300 to the solution and stir gently at 120 rpm for 30 min until fully dissolved. Verify that the pH of the solution is 7.50. If it deviates, readjust the pH to 7.50. Seal and store at 2-8℃. The concentrations of each component in R1 are: 50 mmol / L Na₂HPO₄-KH₂PO₄ buffer (pH 7.5), Tween-20 1.0 ml / L, PEG6000 2 g / L, NaCl 18 g / L, and PC-300 0.2 ml / L.

[0026] 2. Preparation of working microspheres 2.1 Preparation of reagents for the preparation of functional carboxylated latex Latex ①: 0.2 mol / L ethanesulfonic acid (MES), pH 6.50.

[0027] Latex ②: 15 mg / mL N-hydroxysuccinimide (Sulfo-NHS), prepared with 0.2 mol / L pH 6.50 latex ①.

[0028] Latex ③: 4.5 mg / mL carbodiimide (EDC), prepared with 0.2 mol / L pH 6.50 latex ①.

[0029] Latex ④: 0.1 mol / L phosphate buffer, pH 8.25.

[0030] Latex ⑤: 0.1 mol / L phosphate buffer containing 12.5 g / L glycine, pH 8.25.

[0031] Latex ⑥: 0.1 mol / L phosphate buffer containing 200 g / L BSA, pH 8.25.

[0032] (1) Take 0.25 mL of 10% Japanese JSR P0011 latex (100 nm), add 0.05 mL of pH 6.50 latex ① solution, and place in a 37°C constant temperature shaker at 225 r / min for 2 minutes. (2) After removing the solution from the shaker, place it on a magnetic stirrer and, while stirring, add 0.04 mL of pH 6.50 latex solution ② and 0.04 mL of latex solution ③ in sequence. (3) After adding the latex solution ③, place it on a shaker and react for 15 min.

[0033] 2.2 Antibody cross-linking: (4) After the reaction is complete, take it out and add 6.00 mL of latex solution ④ with pH 8.25 while stirring.

[0034] (5) Slowly add 0.40 mg of antibody to the solution and react in a shaker at 225 r / min for 3 hours.

[0035] 2.3, Enclosure: (6) After the reaction is complete, take it out and place it on a magnetic stirrer. While stirring, add 2.00 mL of pH 8.25 latex ④ solution, 0.10 mL of pH 8.25 latex ⑤ solution, and 0.10 mL of pH 8.25 latex ⑥ solution in sequence. Shake at 225 r / min for 1 hour.

[0036] 2.4 Preparation of latex immunoturbidimetric reagent 2 (R2): (7) Place sodium chloride, sucrose, trehalose, bovine serum albumin (BSA), and preservative PC-300 into the above solution, stir gently at 120 rpm for 30 min until fully dissolved, and bring the volume to 10 mL; verify that the pH value of the solution is 8.00. If it deviates, readjust the pH value to 8.00; carry out the aging reaction for 24 hours, seal, and store at 2-8℃. The concentrations of each component in R2 are as follows: ADPN antibody protein functionalized latex microspheres 2.5 g / L, sodium chloride 9 g / L, sucrose 50 g / L, trehalose 20 g / L, PC-300 0.2 ml / L, and BSA 2.5 g / L.

[0037] 3. Sample preparation High-value sample: serum sample with an ADPN concentration of 40 mg / L; Diluent: 0.9% NaCl solution (containing 5 g / L BSA, simulating serum matrix); Linear gradient samples: Linear 1 (0 mg / L): Pure dilution; Linear 2 (2.5 mg / L): 40 mg / L high-value samples were diluted 1:15; Linearity 3 (5 mg / L): 40 mg / L high-value samples were diluted 1:7; Linear 4 (10 mg / L): High-value samples of 40 mg / L were diluted 1:3; Linear 5 (20 mg / L): 40 mg / L high-value samples were diluted 1:1; Linear 6 (40 mg / L): Undiluted high-value sample.

[0038] 4. Measurement Method The Olympus AU600 automated biochemical analyzer was used for the tests. The specific test conditions and procedures are shown in Tables 2 and 3, respectively.

[0039] Table 2 Measurement conditions

[0040] Table 3 Measurement Procedure

[0041] 5. Calibration curve Five-point concentration calibration involves sequentially inputting the concentrations at each point into the instrument. Multi-point calibration is performed using nonlinear spline fitting. The instrument automatically generates a dose-response curve (calibration curve) during calibration, and the sample concentration value is obtained from the calibration curve.

[0042] Example 2: Optimization of EDC Concentration in Crosslinking System The kit was prepared and tested according to the method in Example 1, wherein different concentrations of carbodiimide (EDC) of 3.5, 4.0, 4.5, and 5.0 mg / mL were set in latex ③. The test results are shown in Table 4. Table 4. Absorbance values ​​of calibrators for different EDC concentrations

[0043] Experimental results show that EDC concentrations in the range of 3.5 mg / mL to 5.0 mg / mL exhibit good cross-linking effects, especially at 4.5 mg / mL, where the cross-linking efficiency is the highest, the linearity of the calibration curve is optimal, and the absorbance value is also the largest.

[0044] Example 3: Optimization of Sulfo-NHS concentration in the crosslinking system The kit was prepared and tested according to the method in Example 1, wherein different concentrations of N-hydroxysuccinimide (Sulfo-NHS) of 12, 15, 18, and 21 mg / mL were set in latex ②. The test results are shown in Table 5. Table 5. Optimization of Sulfo-NHS concentration in the crosslinking system

[0045] Experimental results show that the absorbance of the calibration curve gradually increases with increasing Sulfo-NHS concentration, indicating that the efficiency of the crosslinking reaction is improved. In particular, when the Sulfo-NHS concentration is 15 mg / mL, the calibration curve obtained in the experiment has a good fit with the standard curve, indicating that the crosslinking efficiency is ideal at this concentration.

[0046] Example 4: Optimization of latex microsphere concentration in crosslinking system The kit was prepared and tested according to the method in Example 1. For the preparation of the working microspheres, 0.15 mL, 0.20 mL, 0.25 mL, and 0.30 mL of 10% Japanese JSR P0011 latex (100 nm) were used, corresponding to latex microsphere concentrations of 1.5 g / L, 2.0 g / L, 2.5 g / L, and 3.0 g / L in the crosslinking system. The test results are shown in Table 6. Table 6 Optimization of latex microsphere concentration in crosslinking system

[0047] Experimental results show that the concentration of latex microspheres in the crosslinking system significantly affects the morphology of the calibration curve and the reaction sensitivity. At low concentrations (1.5 g / L), the absorbance value generated by the crosslinking reaction is low, and the response of the calibration curve is not obvious, resulting in low detection sensitivity. As the concentration of latex microspheres increases, the absorbance value of the reaction gradually increases, and the linear relationship of the calibration curve becomes more obvious, significantly improving the reaction sensitivity. When the concentration of latex microspheres reaches 2.5 g / L, the absorbance value and the linearity of the calibration curve are optimal, showing the highest crosslinking efficiency and the best detection performance.

[0048] Example 5: Optimization of ADPN antibody concentration in cross-linking system The kit was prepared and tested according to the method in Example 1. In the antibody crosslinking step (5), 0.30 mg, 0.40 mg, 0.50 mg, and 0.60 mg of antibody were slowly added to the solution. The test results are shown in Table 7. Table 7 Optimization of ADPN antibody concentration in cross-linking system

[0049] Experimental results show that different concentrations of ADPN antibody have varying degrees of impact on the absorbance of the calibration curve.

[0050] By comparing the data from each group, it was found that the ADPN antibody concentration of 0.40 mg / 10 mL showed the best performance in terms of calibration curve response, linear range, and test stability. Specifically, the calibration curve showed a high degree of fit, and the absorbance changes at multiple concentration points were quite significant. Therefore, the ADPN antibody concentration of 0.40 mg / 10 mL is the optimal concentration.

[0051] Example 6: Optimization of cross-linking time between cross-linked latex and ADPN antibody The kit was prepared and tested according to the method in Example 1. In the antibody crosslinking step (5), the shaker was set to 225 r / min for 2, 3, 4, and 5 hours respectively. The test results are shown in Table 8. Table 8 Optimization of crosslinking time between latex and ADPN antibody

[0052] The results showed that crosslinking time had a significant impact on crosslinking efficiency. In the crosslinking reactions at 2 and 3 hours, the latex-ADPN antibody complex exhibited better crosslinking performance, with high correlation in the calibration curves and a relatively stable absorbance value trend. The absorbance was highest at 3 hours, indicating the best performance. However, with prolonged crosslinking time (4 and 5 hours), the crosslinking efficiency did not significantly improve, and no effective calibration data was obtained at 5 hours, suggesting that excessively long reactions may lead to loss of antibody activity or overreaction of the crosslinking product.

[0053] Example 7: Optimization of glycine-coated latex particle concentration in crosslinking system The microspheres were prepared and tested according to the method and kit in Example 1. In the preparation of the working microspheres, latex ⑤ was prepared using a 0.1 mol / L phosphate buffer containing 10.0 g / L, 12.5 g / L, 15.0 g / L, and 17.5 g / L glycine. The test results are shown in Table 9. Table 9. Optimization of glycine-coated modified latex particle concentration

[0054] Experimental results show that the reagent's sensitivity varies little and its overall performance is stable within the glycine concentration range of 10.0 g / L to 17.5 g / L. Specifically, the reagent exhibits optimal sensitivity at a glycine concentration of 12.5 g / L, and the sensitivity differences are not significant at other concentrations.

[0055] Example 8: Optimization of BSA-coated latex particle concentration in crosslinking system The kit was prepared and tested according to the method in Example 1. In the preparation of the working microspheres, latex ⑥ was prepared with 0.1 mol / L phosphate buffer containing 200 g / L, 250 g / L, 300 g / L, and 350 g / L BSA. The test results are shown in Table 10 below: Table 10 Optimization of BSA-coated modified latex particle concentration

[0056] Optimization experiments on the concentration of BSA-coated modified latex particles determined the optimal BSA concentration to be 200 g / L. At this concentration, the prepared reagent exhibited good sensitivity and stability. According to the experimental data, the reagent's sensitivity decreased at higher BSA concentrations (250 g / L and above), and the absorbance change in the calibration curve was smaller, indicating that excessively high BSA concentrations may lead to non-specific adsorption or other adverse effects. The 200 g / L BSA concentration showed consistent responses across multiple concentration points and exhibited a good linear fit with the ADPN concentration.

[0057] Example 9: The effect of different buffer solutions on detection results The kit was prepared and tested according to the method in Example 1. Different buffer solutions were used when preparing reagent 1 (R1). The specific components and test results are as follows: Group 1 (PIPES-NaOH buffer): 50 mmol / L PIPES-NaOH buffer (pH 7.00): Weigh 15.12 g of PIPES and dissolve it in 1 L of deionized water. Adjust the pH to 7.00 with 3 mol / L NaOH. Add 0.5 ml / L Tween-80, 4 g / L polyethylene glycol 6000, 9 g / L NaCl, and 0.2 ml / L PC-300. Stir to dissolve and then dispense into containers.

[0058] Group 2 (glycine-NaOH buffer): 50 mmol / L glycine-NaOH buffer (pH 7.00): Weigh 3.75 g of glycine, dissolve it in 1 L of deionized water, and adjust the pH to 7.00 with 3 mol / L NaOH. The remaining components are the same as above.

[0059] Group 3 (Na2HPO4-KH2PO4 buffer): 50 mmol / L Na2HPO4-KH2PO4 buffer (pH 7.00): Weigh 17.91 g of Na2HPO4·12H2O and 1.36 g of KH2PO4, dissolve them in 1 L of deionized water, and adjust the pH to 7.00 with 3 mol / L KOH. The remaining components are the same as above.

[0060] Group 4 (Tris-HCl buffer): 50 mmol / L Tris-HCl buffer (pH 7.00): Weigh 6.06 g of Tris base, dissolve it in 1 L of deionized water, and adjust the pH to 7.00 with 3 mol / L HCl. The remaining components are the same as above.

[0061] Table 11 Absorbance values ​​of calibration curves under different buffer solutions

[0062] The results are shown in Table 11. It can be seen that the Na2HPO4-KH2PO4 buffer performed best among the four groups, with a good curve fit between its absorbance value and ADPN concentration. The Tris-HCl buffer group also showed a good curve fit, but the reaction sensitivity decreased, and the absorbance change in the calibration curve was small. The glycine-NaOH and PIPES-NaOH buffers showed large absorbance changes and poor curve fit due to insufficient pH stability or ion chelation. Therefore, 50 mmol / L Na2HPO4-KH2PO4 buffer (pH 7.00) was selected as the optimal buffer system for R1.

[0063] Example 10: pH Optimization of Phosphate Buffer System The reagent kit was prepared and tested according to the method in Example 1. When preparing reagent 1 (R1), the pH values ​​of the phosphate buffer system were set to 6.00, 6.50, 7.00, 7.50, and 8.00. The test results are shown in Table 12. Table 12 Absorbance values ​​of calibration curves for phosphate buffer systems at different pH values

[0064] Analysis of the results showed that the Na₂HPO₄-KH₂PO₄ buffer system at pH 7.50 performed optimally, with its absorbance value exhibiting a significant gradient change with increasing ADPN concentration (ΔA = 0.4571 at 40 mg / L). The pH 7.00 group was the second best, while the pH 8.00 group showed a significant decrease in sensitivity. Therefore, pH 7.50 was selected as the optimal pH condition for the reaction.

[0065] Example of testing: Validation of reagent kit effectiveness The kit and method prepared in Example 1 were used for testing.

[0066] (1) Limit of Detection (LOD), Limit of Quantification (LOQ) and Linearity Range Test 1.1 Sample Preparation: High-value sample: serum sample with an ADPN concentration of 40 mg / L; Diluent: 0.9% NaCl solution (containing 5 g / L BSA, simulating serum matrix); Linear gradient samples: Linearity 1 (0 mg / L): pure dilution; Linearity 2 (2.5 mg / L): 40 mg / L high-value sample diluted 1:15; Linearity 3 (5 mg / L): 40 mg / L high-value sample diluted 1:7; Linearity 4 (10 mg / L): 40 mg / L high-value sample diluted 1:3; Linearity 5 (20 mg / L): 40 mg / L high-value sample diluted 1:1; Linearity 6 (40 mg / L): undiluted high-value sample.

[0067] 1.2 Experimental Methods 1.2.1 Principle Under the condition of a fixed optimal sample-reagent ratio (3 μL sample, R1 160 μL, R2 40 μL), the blank value b and standard deviation s were calculated using the blank solution repeated detection method. The limit of detection (LOD) and limit of quantitation (LOQ) were determined using the formulas LOD = b + 3s and LOQ = b + 10s. Simultaneously, samples of different concentrations were prepared using high-value mixed serum through serial dilutions (1, 1 / 2, 1 / 4, 1 / 8, 1 / 16). The linear range of the method was evaluated through calibration curves and linear analysis. The absolute deviation was tested to ensure it was ≤ ±0.40 mg / L in the range of [0.38-4.00 mg / L], and the relative deviation was tested to ensure it was within ±10% in the range of [4.00-40.00 mg / L]. The linear correlation coefficient was also calculated.

[0068] 1.2.2 ADPN reagent standard curve test The standard curve was determined according to the method in Example 1.

[0069] Limit of Detection (LOD) and Limit of Quantification (LOQ): Blank values ​​obtained by repeating the detection of blank solution 10 times. b and standard deviation s are used to estimate the limit of detection (LOD) and the limit of quantitation (LOQ). The calculation formula is: LOD = b+3s, LOQ= b+10s.

[0070] Linearity test: Take one high-concentration mixed serum (H) and use 0.9% sodium chloride solution (B) to prepare five concentration gradient mixed serums at ratios of 1 / 2, 1 / 4, 1 / 8, 1 / 16, and 1 / 32. The expected concentration is calculated using the formula X = (CH × VH) / (VB + VH). During testing, each sample is measured in order from low to high concentration. The data are examined according to the protocol provided in CLSI EP6-A (CLSI. Evaluation of the linearity of quantitative measurement procedures: A Statistical Approach; Approved Guideline [S]. CLSI EP6-A. Wayne, PA: CLSI, 2003.). With the measured value as Y and the expected concentration as X, a calibration curve is plotted to obtain the linear equation and correlation coefficient r.

[0071] 1.3 Experimental Results and Conclusions 1.3.1 Limit of Detection (LOD) and Limit of Quantification (LOQ) Table 13 shows the results of the blank solution assay. Table 13

[0072] Calculate the mean b and standard deviation s of the blank values. According to the formula, the blank value mean (b): -0.033, standard deviation (s): 0.041; LOD = b + 3s = -0.033 + 3 × 0.041 = 0.09 mg / L; LOQ = b + 10s = -0.033 + 10 × 0.041 = 0.38 mg / L.

[0073] 1.3.2 Determination of Linear Range One sample of high-concentration mixed serum with a measured concentration of 40 mg / L was used to plot a calibration curve. The results are shown in Table 14 and 15. Figure 1 The calibration curves showed that within the concentration range of 4.00–40.00 mg / L, the relative deviations of all samples were within ±10%; while in the low concentration range (0.38–4.00 mg / L), the absolute deviations were ≤ ±0.40 mg / L. The linear correlation coefficient r ≥ 0.9998, meeting the methodological requirements.

[0074] Table 14 Linearity Test Results

[0075] Experimental results show that the kit provided by this invention has the following characteristics: The LOD is 0.09 mg / L and the LOQ is 0.38 mg / L, which meets the requirements for low concentration detection. The linear range covers 2.50–40.00 mg / L, and the deviations within the commonly used clinical concentration ranges (0.38–4.00 mg / L and 4.00–40.00 mg / L) all meet the technical requirements. The high correlation coefficient of the calibration curve (r ≥ 0.9998) indicates that the method has good quantitative accuracy and repeatability.

[0076] (2) Recovery rate and interference experiment 2.1 Experimental Methods 2.1.1 Recovery rate determination a. Take mixed serum samples and add ADPN standard solutions of low (6.50 mg / L), medium (18.25 mg / L), and high (30.00 mg / L) concentrations respectively to prepare standard mixed serum working solutions; b. Take an equal volume of mixed serum sample and calibrator, and add an equal volume of 0.9% NaCl solution as a control; c. The ADPN concentration of each sample was determined using a fully automated biochemical analyzer (AU400), and the results were repeated three times and the average value was taken. d. Calculate the recovery rate: Recovery rate = [(actual concentration of standard mixed serum working solution – actual concentration of mixed serum) / actual concentration of calibrator] × 100%.

[0077] 2.1.2 Interference Experiment a. Prepare interfering serum containing ascorbic acid (55 mg / dL), conjugated bilirubin (30 mg / dL), hemoglobin (550 mg / dL), and fat emulsion (600 mg / dL) according to the CLSI EP7-A2 guideline (CLSI. Interference Testing in Clinical Chemistry; Approved Guideline. CLSI EP7-A2. Wayne, PA: CLSI, 2005.). b. Use the same volume of 0.9% NaCl solution as a baseline control; c. Measure the concentration of ADPN in each interfering serum, repeat 3 times and take the average value; d. Calculate Diff%: Diff%=[(interference serum measurement value)] [Basic control measurement value) / Basic control measurement value] × 100% 2.2 Experimental Results and Conclusions Recovery rate determination: A mixed serum sample with an ADPN concentration of 5.18 mg / L was taken, and different amounts of ADPN standard solution were added for recovery tests, as shown in Table 15. The recovery rate of ADPN detection ranged from 93.40% to 104.31%, with an average recovery rate of 98.69%. This meets the recovery rate requirements for clinical detection methods (90%-110%).

[0078] Table 15 Serum ADPN detection recovery rate

[0079] The results of the interference experiment are shown in Table 16: Mixed serum samples containing different concentrations of interfering substances were measured, and the presence of interference was determined using ±10% as the standard. For serum detection with an ADPN concentration of 5.55 mg / L, the maximum permissible concentrations of the main interfering substances were: ascorbic acid ≤55 mg / dL, conjugated bilirubin ≤30 mg / dL, hemoglobin ≤550 mg / dL, and fat emulsion ≤600 mg / dL. This indicates that the anti-interference capability meets the requirements of the CLSI EP7-A2 guideline.

[0080] Table 16 Serum ADPN Interference Experiment

[0081] (3) Validation of the HOOK effect 3.1 Experimental Methods 3.1.1 Experimental Principle The HOOK effect refers to the phenomenon where, under high antigen concentration conditions, excess antigen leads to antibody binding site saturation, reducing immune complex formation, and causing the absorbance value (ΔA) to decrease with increasing concentration. This experiment measured the ΔA value at various dilutions of a high-concentration sample (400 mg / L) to observe whether a signal attenuation trend existed, and determined the upper limit of linearity of the detection method according to the CLSI EP17-A guideline (CLSI. Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline. CLSI EP17-A, Wayne, PA: CLSI, 2004.).

[0082] 3.1.2 Specific Measurement a. Prepare diluted samples according to Table 17; b. Use AU400 to determine the ΔA value of each diluted sample; c. Analyze the trend of ΔA with concentration to determine whether the HOOK effect exists.

[0083] Table 17 Absorbance (ΔA) Measurements at Different Dilutions

[0084] 3.2 Experimental Results and Conclusions HOOK effect validation: The results are shown in Table 17. The ΔA value of the undiluted sample (400 mg / L) was 0.3735. After a 2-fold dilution, ΔA increased to 0.4170, and reached a peak of 0.4990 at a 4-fold dilution. Subsequently, ΔA gradually decreased with increasing dilution factor. This indicates that a HOOK effect exists at concentrations ≥400 mg / L, and the optimal upper limit of linearity for detection is 100 mg / L (4-fold dilution).

[0085] Clinical application scope: This method is applicable to the detection of samples with ADPN concentration ≤100 mg / L. For samples exceeding this range, dilution is required before measurement.

[0086] (4) Intra-batch and inter-batch imprecision tests According to the protocol provided in the Clinical and Laboratory Standards Institute (CLSI) EP15-A (CLSI. User demonstration of performance for precision and accuracy, Approved Guideline. CLSI document EP15-A. Wayne, PA: CLSI, 2001.), three mixed serum samples with low, medium, and high levels of chromosomal abnormalities were measured 12 times consecutively within one day to calculate intra-assay imprecision (intra-assay CV), and once daily for 12 consecutive days to calculate inter-assay imprecision (inter-assay CV).

[0087] The experimental results are shown in Tables 18 and 19. The analysis of the results revealed that: Intra-assay imprecision: The CVs for low, medium, and high concentration samples were 2.16%, 1.16%, and 0.78%, respectively, all meeting the CLSI EP15-A requirement for clinical testing methods with a CV ≤ 5%.

[0088] Inter-batch imprecision: The CVs for low, medium, and high concentration samples were 2.60%, 1.91%, and 1.05%, respectively, indicating that the method has good stability under different time and operating conditions.

[0089] Table 18 Intra-batch precision results

[0090] Table 19 Inter-batch precision results

[0091] (5) Accelerated stability study of the reagent kit (stored at 37℃ for 15 days) 5.1 Experimental Objective Based on the Arrhenius accelerated stability equation, the stability changes under long-term storage conditions (8°C) were simulated by placing reagent 1 (R1) and reagent 2 (R2) in a 37°C water bath for 15 days. The accelerated stability of the kit was evaluated, its shelf life was predicted, and the compliance of key performance indicators (absorbance of calibration curve and measured values ​​of quality control materials) was verified, providing experimental basis for the clinical application and storage conditions of the kit.

[0092] 5.2 Reagent Preparation: Calibrator: Take the lyophilized calibrator, accurately add 1 mL of deionized water to reconstitute it, vortex it 3 times, let it stand for 15 minutes before use, and set the value to 39.82 mg / L.

[0093] Quality control products: Take the lyophilized calibrator, accurately add 1 mL of deionized water to reconstitute it, vortex the mixture 3 times, let it stand for 15 minutes before use. The target value of quality control 1 is 3.17±0.32 mg / L, and the target value of quality control 2 is 14.35±1.44 mg / L.

[0094] 5.3 Experimental Methods 5.3.1. Measure the absorbance of the calibration curve at each time point after acceleration (0, 2.49, 4.98, 9.96, 19.92, 39.82 mg / L, a total of 6 points), and calculate the relative deviation of the absorbance value at the 6th point (39.82 mg / L): Relative deviation (%) = (Absorbance after acceleration) (Absorbance on 0 days) / (Absorbance on 0 days × 100%) 5.3.2. Determine the concentrations of quality control sample 1 and quality control sample 2, repeating the process three times at each time point. Calculate the mean, standard deviation (s), imprecision (CV%), and relative deviation from the target value. Relative deviation (%) = (Measurement mean) (Target value) / Target value × 100%.

[0095] 5.3.3 Stability Prediction Model Based on the Arrhenius acceleration stability equation, in this acceleration stability study: Predicted stability = Accelerated stability × 2ΔT / 5 Where ΔT is the difference between the accelerated stabilization storage temperature (37℃) and the upper limit of the storage temperature (8℃). In this study, the sample stabilized for 14 days at 37℃ will be stable for 26 months (14 × 25.8) = 800 days ≈ 26 months at 8℃.

[0096] 5.4 Experimental Results and Conclusions As shown in Table 20, with the extension of acceleration time, the absorbance value at point 6 (39.82 mg / L) gradually decreased, and the relative deviation reached -13.09% at 15 days, exceeding the stability limit of ±10%. The absorbance changes at other concentration points were all within the controllable range (relative deviation ≤ ±5.91%).

[0097] Table 20 Results of accelerated stability test of reagents

[0098] Further analysis results: The relative deviation of the mean value of quality control 1 (3.17 mg / L) was -4.31% (15 days), and the CV% increased to 6.23%; the relative deviation of the mean value of quality control 2 (14.35 mg / L) was -1.30% (15 days), and the CV% was 2.99%. By day 15, the CV% of quality control 1 exceeded the allowable range of 5%, while quality control 2 still met the requirements (CV% < 3%).

[0099] After the reagent was accelerated at 37℃ for 15 days, the relative deviation of absorbance at point 6 of the calibration curve (-13.09%) and the CV% (6.23%) of quality control sample 1 exceeded the limit, indicating that the reagent had failed to maintain stability. The reagent was stable for 14 days when accelerated at 37℃. Based on the prediction of the Arrhenius equation, the shelf life of the kit at 2~8℃ is 26 months.

[0100] (6) Opening stability test 6.1 Experimental Objective To evaluate the stability of the ADPN test kit after opening and storage at 2–8°C, and to determine its effective shelf life after opening, so as to provide a basis for the standardized use of reagents in clinical laboratories.

[0101] 6.2 Material Preparation: Calibrator: Take the lyophilized calibrator, accurately add 1 mL of deionized water to reconstitute it, vortex it 3 times, let it stand for 15 minutes before use, and set the value to 39.82 mg / L.

[0102] Quality control products: Take the lyophilized calibrator, accurately add 1 mL of deionized water to reconstitute it, vortex the mixture 3 times, let it stand for 15 minutes before use. The target value of quality control 1 is 3.17±0.32 mg / L, and the target value of quality control 2 is 14.35±1.44 mg / L.

[0103] 6.3 Experimental Methods 6.3.1 Reagent opening and storage Store the newly prepared ADPN test kits (R1, R2) in the 4℃ reagent compartment of the biochemical analyzer with the cap open to simulate actual usage conditions. After each use, immediately reseal the kit to avoid temperature fluctuations within the reagent compartment.

[0104] 6.3.2 Measurement Time Points and Sample Processing At day 0, take the newly reconstituted calibrator for calibration. No further calibration is performed thereafter. On days 7, 14, 21, 28, and 35 after opening the cap, take the newly reconstituted quality control sample for testing.

[0105] Quality control sample testing: At each time point, quality control sample 1 (3.17 mg / L) and quality control sample 2 (14.35 mg / L) were measured three times, and the mean and relative deviation were calculated. 6.3.3 Evaluation Criteria Repeatability: The CV% of three measurements at the same time point is ≤10%; Accuracy: The relative deviation between the measured mean and the target value is ≤ ±10%. Stability assessment: If all time points meet the above criteria, the reagent is considered to be stable for 35 days after opening.

[0106] 6.4 Experimental Results and Conclusions The test results are summarized in Table 21. After opening the ADPN test reagent, the relative deviation (maximum -9.67%) and CV% (maximum 2.28%) of the quality control test values ​​within 35 days of storage at 2~8℃ both meet the preset standards (±10%, CV≤10%), indicating that the reagent has good stability after opening. It is recommended that the test be completed within 35 days after opening in clinical use to ensure accuracy.

[0107] Table 21 Stability evaluation of ADPN test reagents after opening at 2~8℃

[0108] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A serum adiponectin latex immunoturbidimetry reagent, characterized by, The solution containing ADPN antibody protein functionalized latex microspheres comprises ADPN antibody protein functionalized latex microspheres, a stabilizer, a protective agent, an inorganic salt, and a preservative. The preparation method of the solution containing ADPN antibody protein functionalized latex microspheres comprises the following steps: S1, take 8%-12% of the latex microsphere suspension, add 0.20-0.50 mL of buffer A, and obtain solution 1; S2, add 0.02-0.10 mL of buffer A containing 12-21 mg / mL Sulfo-NHS and 0.02-0.10 mL of buffer A containing 3-6 mg / mL EDC to solution 1, and perform activation reaction to obtain solution 2; S3, add 4.0-10.0 mL of buffer B to solution 2, add ADPN antibody, and perform antibody and microsphere coupling reaction to obtain solution 3; S4, add 0-3 mL of buffer B, 0.05-0.30 mL of buffer B containing 10.0-17.5 g / L glycine, and 0.05-0.30 mL of buffer B containing 150-350 g / L BSA to solution 3, and perform blocking reaction to obtain solution 4; S5, add inorganic salt, protective agent, stabilizer, and preservative to solution 4, and perform aging reaction to obtain the solution containing ADPN antibody protein functionalized latex microspheres; The buffer A is selected from any one or more of phosphate buffer, MES buffer, MOPS buffer, glycine buffer, Tris buffer, or HEPES buffer; The buffer B is selected from any one or more of phosphate buffer, MES buffer, MOPS buffer, glycine buffer, Tris buffer, or HEPES buffer.

2. The serum adiponectin latex immunoturbidimetry reagent according to claim 1, characterized by, The preparation method comprises the following steps: S1, take 8%-12% of the 90-110 nm latex microsphere suspension, add 0.20-0.50 mL of buffer A, and shake well at 35-40°C to obtain solution 1; S2, under stirring, add 0.02-0.10 mL of buffer A containing 12-21 mg / mL Sulfo-NHS and 0.02-0.10 mL of buffer A containing 3-6 mg / mL EDC to solution 1, and perform activation reaction for 10-30 min to obtain solution 2; S3, under stirring, add 4.0-10.0 mL of buffer B to solution 2, add 0.3-0.6 mg of ADPN antibody, and perform antibody and microsphere coupling reaction for 2-5 hours to obtain solution 3; S4, under stirring, add 1-3 mL of buffer B, 0.05-0.30 mL of buffer B containing 10.0-17.5 g / L glycine, and 0.05-0.30 mL of buffer B containing 150-350 g / L BSA to solution 3, and perform blocking reaction for 1-3 hours to obtain solution 4; S5, adding inorganic salt, protective agent, stabilizer, preservative to solution 4, adjusting pH to 7.0-9.0, aging reaction for 24-72 hours, obtaining solution containing ADPN antibody protein functionalized latex microspheres.

3. The serum adiponectin latex immunoturbidimetry reagent according to claim 2, characterized by, The preparation method comprises the following steps: S1, taking 10% of 100 nm latex microspheres suspension, adding 0.25 mL of buffer A, shaking and mixing at 37℃, obtaining solution 1; S2, adding 0.04 mL of buffer A containing 12-21 mg / mL Sulfo-NHS and 0.04 mL of buffer A containing 3-6 mg / mL EDC to solution 1 under stirring, activating reaction for 15 min, obtaining solution 2; S3, adding 6.0 mL of buffer B to solution 2 under stirring, adding 0.3-0.6 mg of ADPN antibody, coupling reaction of antibody and microspheres for 2-5 hours, obtaining solution 3; S4, adding 2 mL of buffer B, 0.10 mL of buffer B containing 10.0-17.5 g / L glycine and 0.10 mL of buffer B containing 150-350 g / L BSA to solution 3 under stirring, blocking reaction for 1 hour, obtaining solution 4; S5, adding inorganic salt, protective agent, stabilizer, preservative to solution 4, adjusting pH to 8.0, aging reaction for 24 hours, obtaining solution containing 1.5-3.0 g / mL ADPN antibody protein functionalized latex microspheres.

4. The serum adiponectin latex immunoturbidimetry reagent according to claim 3, characterized in that, The buffer A in step S1 is 0.1-0.3 mol / L of MES buffer, preferably 0.2 mol / L of MES buffer; The concentration of Sulfo-NHS in buffer A in step S2 is 15 mg / mL, and the concentration of EDC in buffer A is 4.5 mg / mL; The buffer B in step S3 is 0.05-0.20 mol / L of phosphate buffer, preferably 0.10 mol / L of phosphate buffer; The mass of ADPN antibody added in step S3 is 0.4 mg, and the coupling reaction is carried out on a shaking table for 3 hours; The concentration of glycine in buffer B in step S4 is 12.5 g / L, and the concentration of BSA in buffer B is 200 g / L; The final concentration of ADPN antibody protein functionalized latex microspheres in solution in step S5 is 2.5 g / mL.

5. The serum adiponectin latex immunoturbidimetry reagent according to any one of claims 1-4, characterized in that, The protective agent is selected from any one or more of BSA, mannitol, chitosan or casein, preferably BSA; The stabilizer is selected from any one or more of sucrose or trehalose; The inorganic salt is selected from any one or more of magnesium chloride, sodium chloride, potassium chloride, calcium chloride, aluminum chloride, preferably sodium chloride; The preservative is selected from any one or more of Proclin 300, sodium azide, phenol, gentamicin or thiomersal, preferably Proclin 300. 6.The serum adiponectin latex immunoturbidimetry reagent of claim 5, characterized in that, the concentration of the protective agent is 5-100 g / L, the concentration of the stabilizer is 1.0-20.0 g / L, the concentration of the inorganic salt is 1.0-20.0 g / L, and the concentration of the preservative is 0.01-3.0 g / L.

7. The serum adiponectin latex immunoturbidimetry reagent according to any one of claims 1 to 4, characterized by, The reagent 1 further comprises a buffer, a surfactant, a coagulant, an inorganic salt and a preservative. The buffer is selected from any one or more of citrate buffer, phosphate buffer, carbonate buffer, glycine buffer, Tris buffer or MES buffer, preferably phosphate buffer. The surfactant is selected from any one or more of Tween-20, Tween-80 or TritonX-100, preferably Tween-20. The coagulant is selected from any one or more of polyethylene glycol 8000, polyethylene glycol 6000 or polyethylene glycol 2000, preferably PEG6000. The inorganic salt is selected from any one or more of magnesium chloride, sodium chloride, potassium chloride, calcium chloride or aluminum chloride, preferably sodium chloride. The preservative is selected from any one or more of Proclin300, sodium azide, phenol, gentamicin or thiomersal, preferably Proclin300. 8.The serum adiponectin latex immunoturbidimetry reagent of claim 7, characterized in that, the concentration of the buffer is 10-300 mM, the concentration of the surfactant is 0.2-20.0 g / L, the concentration of the coagulant is 0.5-5.0 g / L, the concentration of the inorganic salt is 10-50 g / L, and the concentration of the preservative is 0.01-3.00 g / L. 9.The serum adiponectin latex immunoturbidimetry reagent of any one of claims 1-8 for use in ADPN detection.

10. A method for detecting ADPN, characterized by, The method is detection using the serum adiponectin latex immunoturbidimetry reagent of any one of claims 1-8.

Images